PROJECT SUMMARY Reactive oxygen species (ROS), such as H2O2 derived from NADPH oxidase (NOX) act as signaling molecules to promote VEGF-induced angiogenesis in ECs and post-ischemic neovascularization. Fundamental question remains ?how diffusible H2O2 signal can be efficiently transmitted to promote therapeutic angiogenesis.? Signaling function of ROS is through oxidation of reactive Cys residues to generate ?Cysteine sulfenic acid (Cys- OH)? which is involved in disulfide bond formation and redox signaling. Protein Disulfide Isomerase (PDI) functions as oxidase, reductase and isomerase depending on redox environment. ?PDIA1? is a major PDI isoform with four reactive Cys residues in redox active domains. Given redox properties of PDI, PDI may function as redox sensor in ROS-dependent VEGF signaling to enhance therapeutic angiogenesis and maintain endothelial metabolic states. Preliminary Data found that PDIA1+/- mice or diabetes mice with reduced PDIA1 expression show impaired reparative angiogenesis, indicating in vivo significance of PDIA1. In primary ECs, VEGF stimulation increases Cys-OH formation of various proteins, which was markedly decreased by PDIA1 siRNA. Experiments using 2D gel assay and searching for binding partner of PDIA1 discovered that PDIA1 functions as a redox sensor in ROS-dependent VEGF signaling to promote Cys oxidation/activation of AMPK, a key regulator of cell metabolism and angiogenesis, via disulfide bond formation. Moreover, in quiescent basal ECs, PDIA1 knockdown unexpectedly induced mitochondrial fragmentation and EC senescence without inducing ER stress via increasing Cys oxidation of Drp1, a key fission GTPase. We thus hypothesize that PDIA1 functions as key redox adaptor/reductase for Drp1 to maintain mitochondrial dynamics in quiescent ECs as well as redox sensor when it is Cys oxidized to transduce VEGF-induced H2O2 signal to promote oxidative activation of AMPK via disulfide bond formation, thereby enhancing endothelial metabolism and angiogenesis in ECs. This is required for full neovascularization in ischemic vascular disease. Aim 1 will determine the molecular mechanisms by which PDIA1 senses VEGF-induced H2O2 signal to promote EC metabolism and angiogenesis via oxidative activation of AMPK, which is impaired in diabetic ECs. Aim2 will examine whether PDIA1 maintains mitochondrial dynamics via binding to Drp1 to keep it in reduced/inactive state in quiescent ECs, thereby preventing mitochondrial fragmentation and ECs dysfunction in diabetes. Aim 3 will determine the in vivo role of endothelial PDIA1 in ROS-dependent reparative neovascularization, which is impaired in diabetes. We will use biotin-labelled Cys-OH trapping probe; BiFC-based molecular protein interaction imaging; mitochondrial dynamics imaging; EC-specific PDIA1-/- or diabetic mice; and gene transfer of EC-targeted Cys oxidation defective mutants of PDIA1, AMPK and Drp1. Our proposal will provide novel insights into Cys reduced/oxidized proteins and Cys oxidation-mediated molecular interaction as potential therapeutic targets for treatment of ischemic cardiovascular metabolic diseases.